Summary: Principle Objectives of the Current Research 1. Identification of Bacillus anthracis genes that are essential for survival in human macrophages. 2. Identification of Bacillus anthracis virulence genes that are induced upon entry into the host. 3. Identification of Bacillus anthracis virulence genes that are essential for survival following experimental infection 4. Development of an animal model of B. anthracis infection. Bacillus anthracis is a Gram (+) rod. The development of 1-2 micron endospores that are resistant to temperature and drying provide the bacterium with a dormant state in order to survive within the soil. Infection is thought to occur by inhalation, abrasion or ingestion. Most commonly, anthrax is diagnosed as a cutaneous infection in those with occupational contact with animals or animal products. Cutaneous anthrax is readily curable using antibiotics and rarely progresses to systemic infection (Dixon et al. 1999). In contrast, a systemic infection (usually resulting from inhalation of anthrax spores) has a mortality rate approaching 100%. Following inhalation, spores are phagocytosed by alveolar macrophages, triggering germination to the vegetative form. Viable cells are released into the lymphatic system and later into the bloodstream, causing massive bacteraemia of between 107 to 108 cfu/ml. The vegetative form of B. anthracis produces a number of virulence factors including specific toxins. The production of an acute cytokine response contributes to hypotension, septic shock and death. After testing a number of transposon delivery systems used in a variety of Bacillus and Lactobacillus species, we identified a mini-Tn10 transposon system that we have used successfully to generate transposon insertion mutations in the B. anthracis chromosome. Using this transposon mutagenesis system, we have generated a library of mutagenized B. anthracis. In order to identify B. anthracis transposon mutants affected for their ability to survive within human macrophages, we developed in vitro screens for resistance to killing by human macrophages. To date we have identified five unlinked, chromosomal transposon insertions that result in the loss of resistance to killing by macrophages. The chromosomal location of each transposon insertion has been identified and confirmed by sequence analysis. Each of the open reading frames likely to be affected by each transposon insertion will be individually knocked out and the phenotype of the resulting strains will be assessed using in vitro human macrophage invasion assays. An important aspect of this work will be the generation of unmarked, in-frame deletions of each of the open reading frames of interest. The lack of a functional allele replacement system for B. anthracis has required researchers in the field to rely on Campbell-insertion of entire plasmids in order to generate knock-out mutations. We have successfully tested a conditionally-lethal genetic marker as well as a temperature-sensitive plasmid origin of replication in B. anthracis and we are in the process of combining these elements to construct an allele-replacement plasmid. This plasmid should allow us to generate unmarked, in-frame deletions of defined open reading frames in the B. anthracis. Although our current methods for screening mutants are labor intensive, they continue to yield new loci required for survival of B. anthracis within human macrophages. Until we begin to isolate insertions in previously identified loci, without identifying new loci, it is reasonable to conclude that our mutagenesis and screening system will continue to be productive. Therefore, in the upcoming year, we will continue to utilize our current screens to screen our existing library of B. anthracis transposon mutants for additional insertion mutants affected for their ability to resist killing by macrophages. In order to study the early steps of B. anthracis infection, it will be necessary to utilize an animal model of infection that mimics, as much as possible, the relevant infection in humans. The route of B. anthracis infection that is of most concern is the aerosol route. Therefore, we have undertaken the development of an animal aerosol-challenge model of B. anthracis infection. The injection of A/J mice with the Sterne I (pX01+, pX02-) strain of B. anthracis leads to a systemic infection resulting in death (14). Aerosol exposure of A/J mice with the Sterne I strain has been used successfully to demonstrate vaccine efficacy and therefore should provide a useful model of B. anthracis infection (3). The appropriate dose of B. anthracis spores to be used in these studies will be optimized during initial experiments. In addition, endpoints will be evaluated to determine which are most relevant for the determination of virulence. Those endpoints under consideration include percent survival, time to death, and the number of bacterial colony-forming units isolated from host tissues (lungs, lymph nodes, liver, spleen, and blood) at various timepoints post-infection. The establishment of a mouse aerosol challenge model of B. anthracis infection will allow for the assessment of the contribution of putative virulence factors to the pathogenesis of B. anthracis and will be useful for the evaluation of new vaccine and therapeutic candidates. The development of this model will also make it possible to employ the powerful in vivo genetic systems used in other pathogenic systems for the identification of genes required for infection of the host such as in vivo expression technologies (IVET) and signature-tagged mutagenesis (STM). The IVET "promoter trap" strategy provides a method to identify genes that are induced upon host entry or when B. anthracis is phagocytosed by a macrophage. In contrast, STM provides a method to identify genes that are necessary for the survival of the pathogen during the infective process. The rationale for the use of both the IVET and STM methods is based on observations that these two systems are complementary, and have been shown to yield different results.